Figure 2. Inactivation of Braf in limb muscle precursor cells.
(A) Strategy for generation of Braf floxed mice (Brafnfl). Breeding with MeuCre mice yielded Braffl mice, in which the neomycin cassette was removed but exon three is flanked by loxP-sites. (B) Braffl mice were bred with Pax3-Cre mice to generate animals lacking Braf in Pax3 expressing cells. Pax3-Cre//Brafdel/del embryos die around E15.5. Mutant embryos were analyzed between E10.5 and E14.5. (C) RT-PCR analysis of Met and Pax3 expression in limb buds of Pax3-Cre//Brafdel/del embryos at E10.5 n = 2. (D) Western blot analysis of expression of different markers in limb buds of Pax3-Cre//Brafdel/del embryos at E10.5 and E14.5 n = 2. (E) Expression of WT and CA Braf (VE600E) in C2C12 cells increases expression of Met and Pax3. n = 3. (F) BRAF enhances PAX3-dependent transcriptional responses. The pGl.3 Pax3BS luc reporter construct containing two PAX3 binding sites in front of a minimal promoter was co-transfected with different combinations of Pax3, Braf, and CA Braf (V600E) expression vectors into HEK293T cells. The activity of Firefly Luciferase was normalized by a co-transfected renilla luciferase in all experiments. Data represent the mean ± SEM and analyzed using ANOVA with a Tukey-Kramer post-hoc comparison test. ***p<0.001.